PROJECT SUMMARY The bacterium Pseudomonas aeruginosa is a common opportunistic human pathogen that causes life- threatening illnesses, including acute pneumonia, long-term lung colonization in most cystic fibrosis patients, and severe wound infections, especially in hospitalized patients and those with severe burns. Most P. aeruginosa infections are associated with compromised host defense and this, together with the common environmental occurrence of the organism, makes it a frequent cause of sepsis in the intensive care unit. The resolution of P. aeruginosa disease is challenging, in part because of its intrinsic resistance to antibiotics as well as occasional outbreaks of multi-drug-resistant strains in hospitals. Therefore, there is an urgent need to identify new targets for therapeutic attack. The cell envelope of P. aeruginosa has two C-terminal processing proteases or CTPs (named CtpA and Prc), both of which have been linked to systems associated with disease. In fact, many bacterial CTPs have been linked to virulence but very little is known about the underlying mechanisms. In P. aeruginosa, CtpA is essential for protein export by a machine known as a type 3 secretion system, one of the most important virulence systems for acute infections. Prc has been linked to a regulatory system that controls the production of a surface molecule known as alginate, which is associated with a poor prognosis in cystic fibrosis patients. We have now discovered that CtpA forms a complex with an uncharacterized outer membrane lipoprotein, which facilitates its activity. This proteolytic complex targets at least one enzyme that modifies the bacterial cell wall. We hypothesize that many other bacterial CTPs, including Prc in P. aeruginosa, will also work with partner proteins to target cell wall modifying enzymes, which makes an understanding of how these proteolytic complexes function broadly significant. Therefore, in this work we will: (1) Analyze how the lipoprotein partner facilitates CtpA-dependent proteolysis; (2), Investigate the impact of a CtpA substrate on the cell wall and virulence, and how its proteolysis is controlled within the bacterial cell; (3) Broaden the impact of this work by characterizing additional CtpA substrates and testing our hypothesis that Prc functions similarly. We predict that CTPs will emerge as a conserved mechanism by which bacteria regulate some of their cell wall modifying enzymes. Therefore, not only will this work provide insight into how CTPs function, which will be of relevance to many pathogens, but it will also shed light on how bacteria control the metabolism of one of their most clinically important components, the cell wall. Finally, the accessible cell envelope proteins that we characterize here could eventually be considered as new targets for therapeutic drugs.